Noninvasive, nuclear imaging techniques can be used to obtain basic and diagnostic information about the physiology and biochemistry of a variety of living subjects including experimental animals, normal humans and patients. These techniques rely on the use of sophisticated imaging instrumentation which is capable of detecting radiation emitted from radiotracers administered to such living subjects. The information obtained can be reconstructed to provide planar and tomographic images which reveal distribution of the radiotracer as a function of time. Use of appropriately designed radiotracers can result in images which contain information on the structure, function and most importantly, the physiology and biochemistry of the subject. Much of this information cannot be obtained by other means. The radiotracers used in these studies are designed to have defined behaviors in vivo which permit the determination of specific information concerning the physiology or biochemistry of the the subject or the effects that various diseases or drugs have on the physiology or biochemistry of the the subject. Currently, radiotracers are available for obtaining useful information concerning such things as cardiac function, myocardial blood flow, lung perfusion, liver function, brain blood flow, regional brain glucose and oxygen metabolism.
Compounds can be labeled with either positron or gamma emitting radionuclides. For imaging, the most commonly used positron emitting radionuclidesare .sup.11 C, .sup.18 F, .sup.15 O and .sup.13 N, all of which are accelorator produced, and have half lifes of 20, 110, 2 and 10 min. respectively. Since the half-lives of these radionuclides are so short, it is only feasible to use them at institutions which have an accelerator on site for their production, thus limiting their use. Several gamma emitting radiotracers are available which can be used by essentially any hospital in the U.S. and in most hospitals worldwide. The most widely used of these are .sup.99m Tc, .sup.201 Tl, and .sup.123 I. .sup.123 I is particularly useful as a radiotracer for imaging applications because of its ability to form covalent bonds with carbon which, in many cases, are stable in vivo and which have well-understood effects on physiochemical properties of small molecules.
In the past decade, one of the most active areas of nuclear medicine research has been the development of receptor imaging radiotracers. These tracers bind with high affinity and specificity to selective hormone receptors and neuroreceptors. Successful examples include radiotracers for imagining the following receptor systems: estrogen, muscarinic, dopamine D1 and D2, and opiate.
Currently available chemotherapeutic drugs for treating neoplastic diseases act by disrupting fundamental mechanisms concerned with cell growth, mitotic activity, differentiation and function. The capacity of these drugs to interfere with normal mitosis and cell division in rapidly proliferating tissues is the basis for their therapeutic application as well as toxic properties. As a result, clinical doses of antitumor drugs are a compromise between efficacy and toxicity such that therapeutic doses are usually set close to the toxic levels in order to maximize efficacy. In a similar manner, dose selection for clinical evaluation of new antitumor drugs is a function of the toxicity of the drug where doses used in Phase II and III trials are often the maximally tolerated doses.
The Ras proteins (Ha-Ras, Ki4a-Ras, Ki4b-Ras and N-Ras) are part of a signalling pathway that links cell surface growth factor receptors to nuclear signals initiating cellular proliferation. Mutated ras genes (Ha-ras, Ki4a-ras, Ki4b-ras and N-ras) are found in many human cancers, including colorectal carcinoma, exocrine pancreatic carcinoma, and myeloid leukemias. The protein products of these genes are defective in their GTPase activity and constitutively transmit a growth stimulatory signal.
At least 3 post-translational modifications are involved with Ras membrane localization, required for normal and oncogenic function, and all 3 modifications occur at the C-terminus of Ras. The Ras C-terminus contains a sequence motif termed a "CAAX" or "Cys-Aaa.sup.1 -Aaa.sup.2 -Xaa" box, which, depending on the specific sequence, serves as a signal sequence for the enzymes farnesyl-protein transferase or geranylgeranyl-protein transferase, which catalyze the alkylation of the cysteine residue of the CAAX motif with a C.sub.15 or C.sub.20 isoprenoid, respectively. (S. Clarke., Ann. Rev. Biochem. 61:355-386 (1992); W. R. Schafer and J. Rine, Ann. Rev. Genetics 30:209-237 (1992)). The Ras protein is one of several proteins that are known to undergo post-translational farnesylation. Other farnesylated proteins include the Ras-related GTP-binding proteins such as Rho, fungal mating factors, the nuclear lamins, and the gamma subunit of transducin. James, et al., J. Biol. Chem. 269, 14182 (1994) have identified a peroxisome associated protein Pxf which is also farnesylated. James, et al., have also suggested that there are farnesylated proteins of unknown structure and function in addition to those listed above.
Inhibition of farnesyl-protein transferase has been shown to block the growth of Ras-transformed cells in soft agar and to modify other aspects of their transformed phenotype. Recently, it has been shown that an inhibitor of farnesyl-protein transferase blocks the growth of ras-dependent tumors in nude mice (N. E. Kohl et al., Proc. Natl. Acad. Sci U.S.A., 91:9141-9145 (1994) and induces regression of mammary and salivary carcinomas in ras transgenic mice (N. E. Kohl et al., Nature Medicine, 1:792-797 (1995).
Farnesyl transferase inhibitors (FTIs) represent a new pharmacological approach to the treatment of cancer that is mechanism-based and does not rely on a cytotoxic mechanism of action. Ideally, therapeutically effective doses of FTIs will not be limited by cytotoxic side effects and these compounds will have a much larger therapeutic window than currently available antitumor drugs.
Farnesyl-protein transferase inhibitors may also be useful for inhibiting other proliferative diseases, both benign and malignant, wherein Ras proteins are aberrantly activated as a result of oncogenic mutation in other genes (i.e., the Ras gene itself is not activated by mutation to an oncogenic form) with said inhibition being accomplished by the administration of an effective amount of the instant composition to a mammal in need of such treatment. For example, a component of NF-1 is a benign proliferative disorder.
Use of farnesyl-protein transferase inhibitors in the prevention of restenosis after percutaneous transluminal coronary angioplasty by inhibiting neointimal formation has recently been described (C. Indolfi et al. Nature medicine, 1:541-545(1995)). It has been disclosed that farnesyl-protein transferase inhibitors may also be useful in the treatment and prevention of polycystic kidney disease (D. L. Schaffner et al. American Journal of Pathology, 142:1051-1060 (1993) and B. Cowley, Jr. et al. FASEB Journal, 2:A3160 (1988)).
It has recently been reported that farnesyl-protein transferase inhibitors are inhibitors of proliferation of vascular smooth muscle cells and are therefore useful in the prevention and therapy of arteriosclerosis and diabetic disturbance of blood vessels (JP H7-112930).
In contrast to cytotoxic chemotherapeutic agents, a more rational approach for estimating clinically-effective doses can be used with FTIs, i.e. it may not be necessary to titrate doses in a patient until toxic effects are observed if that dose is considerably higher than needed to provide the desired effect on tumor growth. Alternatively, if dose-limiting toxicity is observed with the clinical FTI, the dose may not be sufficient to inhibit the enzyme to the extent needed to block tumor growth. Demonstration of clinical efficacy using inhibition of tumor growth or regression in tumor size as an endpoint will require considerable time (weeks to months) and, therefore, dose selection for these trials is very important. Plasma drug concentrations are often used for clinical dose selection, however this endpoint may be a poor surrogate for the drug concentration at the pharmacological target, especially when the site of action is intracellular, such as is the case with farnesyl transferase. PET (Positron Emission Tomography) radiotracers and imaging technology may provide a powerful method for clinical evaluation and dose selection of FTIs. Using a carbon-11 or fluorine-18 labeled radiotracer that enters cells and provides a farnesyl transferase (FPTase) enzyme-specific image in tumors and other tissues, the dose required to saturate FPTase can be determined by the blockade of the PET radiotracer image in humans. The rationale for this approach is as follows: anti-tumor efficacy of FTIs is a consequence of the extent of enzyme inhibition, which in turn is a function of the degree of drug-enzyme occupancy.
It is, therefore, an object of this invention to develop radiolabeled farnesyl-protein transferase inhibitor compounds that would be useful not only in traditional imaging applications, but would also be useful in assays, both in vitro and in vivo, for labeling the enzyme and for competing with unlabeled farnesyl-protein transferase inhibitors (FTIs). It is a further object of this invention to develop novel assays which comprise such radiolabeled compounds.
It is also the object of this invention to provide for a radiolabeled farnesyl-protein transferase inhibitor compound which is optimized for in vivo imaging and is therefore useful for determining the appropriate clinical doses of an FTI which will be used to assess antitumor efficacy in humans.